There are many applications in the field of chemical processing in which it is desirable to separate chemical components prior to or after reacting chemicals. Examples of reactions requiring separation of components include organic, inorganic, biochemical, and molecular reactions. Examples of chemical reactions include thermal cycling amplification, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), isothermal nucleic acid amplification, self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, affinity binding assays, and more complex biochemical mechanistic studies. Conventional separation techniques include electrophoresis, such as capillary electrophoresis, synchronized cyclic electrophoresis, and free flow electrophoresis. Conventional separation techniques also include isoelectric focusing (IEF), hybridization, liquid and gas chromatography, molecular sieving and filtering.
Components to be separated in various samples include nucleic acids, amino acids, peptides, proteins, cells, viruses, bacteria, organic compounds, carbohydrates, etc. For example, in amplification applications, multiple oligonucleotide primers and probes designed for many organisms can be used to multiply DNA from numerous organisms in a sample. After amplification, separation techniques such as electrophoresis or IEF can be employed to separate the amplification products by certain properties, such as molecular weight, for subsequent detection by fluorescence methods.
Of increasing interest in the field of chemical separation is the use of devices that include an integrated reaction chamber and separation region. Such integrated devices provide a number of advantages over conventional devices in which one transfers a fluid sample between a reaction apparatus and a separation device. For example, where the chemical reaction and separation steps are performed in a single integrated device, one may avoid contamination and crossover of sample or reaction products. In addition, an integrated device may allow for substantially faster sample processing and analysis.
Recent efforts to integrate processing and analytical functionalities in a single device, especially in the field of MEMS, microfabrication, and microfluidics, have resulted in the development of devices that include multiple substrates bonded together. The substrates are usually bonded with adhesives, or by heat sealing, fusion bonding, or anodic bonding. These multi-substrate devices typically include a reaction chamber that is connected to a separate separation component, such as a capillary tube containing a suitable electrophoresis gel, by an adhesive such as epoxy. Alternatively, these multi-substrate devices have reaction chambers and separation channels etched into a plate and a cover bonded over the top of the plate. For example, U.S. Pat. No. 5,849,208 issued to Hayes et al. discloses such a multi-substrate device.
Unfortunately, prior integrated devices provide for only limited control of fluid between reaction and separation regions. For example, high internal pressure can develop in a reaction chamber due to the thermal expansion of liquid or gas present in this region, the generation of gas bubbles, or the chemical reactions performed inside of the chamber. This pressure, combined with any elevated temperatures within the chamber, can have detrimental effects on fluidic components and performance upstream and downstream from the reaction chamber. A particular problem is the flow or diffusion of chemicals from the reaction chamber into unwanted regions caused by the elevated pressure or temperature. This situation is especially problematic when sensitive detection methods and apparatus are located downstream from the reaction chamber.
A further problem with prior integrated devices is that when the reaction chamber is heated to perform a chemical reaction, the separation region is also heated due to thermal conduction. If the separation region is heated, however, the separation material contained in the region, e.g., electrophoresis gel, degrades and renders the device inoperable. In addition to degrading the separation material, the thermal conduction between the reaction chamber and separation region causes large thermal gradients within the device and prevents adequate heating of a sample in the reaction chamber.
Moreover, where the separation region requires electrodes, as in capillary electrophoresis, micro-electrophoresis, and IEF, there are many issues that have not been addressed by prior art designs. Much of the prior art has focused on issues regarding the design, fabrication, and operation of the capillary channel itself. In contrast, the design of the electrodes which are in contact with the fluid, and which are responsible for the electrokinetic movement of the fluid, has not yet been adequately addressed. The correct design of these electrodes is necessary for the proper, practical, and cost-effective implementation of high-volume, disposable systems that incorporate both reaction chambers and separation regions in a single device. Current state-of-the-art is to incorporate large, open reservoirs (100 μL or more) near the end of each capillary channel, and then “dip” a separate metal electrode into each open reservoir. This arrangement may be unsuitable for mass production of disposable assemblies. Furthermore, external electrodes, which are placed in contact with the fluid in the disposable assembly, are generally not practical because they may need to be cleaned or otherwise “prepared” prior to each use.